Guild proposes to develop an antisense oligomer-based antifungal effective against the human pathogen Aspergillus fumigatus. With a 40%-60% mortality rate, invasive aspergillosis (IA) due to A. fumigatus now surpasses invasive candidiasis as the most frequent fungal cause of death, especially amongst immunocompromised patients. Successful therapy for IA with current antifungals is notoriously difficult due to poor clinical efficacy and the increasing emergence of drug resistant strains. Despite this acute need for novel therapeutic strategies, there has not been a new class of antifungals targeting A. fumigatus developed in over 12 years. Therefore, a novel approach is urgently needed to develop new anti-Aspergillus targeting strategies. Phosphorodiamidate morpholino oligomers (PMOs), synthetic uncharged analogs of nucleic acids, are an antisense biotechnology that functions by base pairing with target gene mRNA, producing steric blockade of the translational machinery. PMOs have been investigated as antimicrobials against bacteria, but applications to fungal pathogens are rare. Our team has recently developed PMOs conjugated to cell penetrating peptides (CPPs) that inhibit the growth of the fungal pathogen Candida albicans. Calcineurin is a highly conserved protein phosphatase that is important in mediating cell stress responses. Research by our team has established that calcineurin is necessary for A. fumigatus growth, and its deletion produces numerous nonlethal defects. Inhibition of calcineurin halts invasive fungal disease. As proof of concept, a CPP-PMO will be developed against the A. fumigatus calcineurin A (CnaA) gene and tested In Vitro for pathway disruption and growth inhibition through the following specific aims. Aim 1. Identify a cell penetrating peptide that optimally accumulates within A. fumigatus and has minimal toxicity to human cells. A set of CPPs from the literature will be compared to the CPP developed against C. albicans. The top accumulating peptide will be optimized through competitive targeted peptide libraries, and then be tested for preliminary off-target toxicity in relevant human cell lines. Aim 2. Demonstrate successful knockdown of A. fumigatus CnaA using a CPP-PMO construct. A PMO will be designed against the start site of the cnaA gene and be conjugated to the CPP from Aim 1. The CPP- PMO will be tested on cultured A. fumigatus with endpoints of CnaA protein knockdown, downstream effector modulation, growth reduction, and off target toxicity. The CnaA inhibiting drug FK506 will be used as a positive CnaA inhibition control. Impact: This project will produce a CPP-PMO that disrupts A. fumigatus growth. In the Phase 2 project, we will evaluate CPP-PMOs against CnaA and other gene targets both alone and in combination with existing antifungal agents in immunosuppressed murine models of IA. This novel approach targeting a critical well- validated A. fumigatus virulence factor will significantl impact the future treatment paradigm of IA.